WO2018014008A1 - Compositions et procédés pour la vaccination contre les alphavirus - Google Patents
Compositions et procédés pour la vaccination contre les alphavirus Download PDFInfo
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- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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- C12N2770/00011—Details
- C12N2770/36011—Togaviridae
- C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
- C12N2770/36122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2770/00011—Details
- C12N2770/36011—Togaviridae
- C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
- C12N2770/36134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the deletion in the E l gene region, the E2b gene region, the E3 gene region, the E4 gene region, or any combination thereof results from a translocation of two or more base pairs.
- the deletion in the E l gene region, the E2b gene region, the E3 gene region, the E4 gene region, or any combination thereof each comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or at least 150 base pairs.
- the deletion in the E 1 gene region, the E2b gene region, the E3 gene region, the E4 gene region, or any combination thereof each comprises more than 150, more than 160, more than 170, more than 180, more than 190, more than 200, more than 250, or more than 300 base pairs.
- the alphavirus target antigen comprises an antigen of a virus selected from the group consisting of Chikungunya virus (CHIKV), o'nyong-nyong virus (ONNV), Ross River virus (RRV), Mayaro fever virus (MAYV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalomyelitis virus (WEEV), and Eastern equine encephalitis virus (EEEV), or any combination thereof.
- the alphavirus target antigen comprises an antigen of a virus selected from the group consisting of CHIKV, ONNV, RRV, and MAYV, or any combination thereof.
- the alphavirus target antigen comprises an antigen of CHIKV.
- the sequence encoding an alphavirus target antigen comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 , or any combination thereof.
- the sequence encoding an alphavirus target antigen is an amino acid sequence, and wherein the amino acid sequence comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 1 1 , and SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 21or any combination thereof.
- the sequence encoding an alphavirus target antigen comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3, or any combination thereof.
- the replication defective virus vector further comprises an element to increase an expression of the alphavirus target antigen.
- the element comprises at least one element, at least 2 elements, at least 3 elements, at least 4 elements, or at least 5 elements.
- the present disclosure provides a pharmaceutical composition comprising the composition of any one of claims 1-34 and a pharmaceutically acceptable carrier.
- the present disclosure provides a cell comprising any one of the above compositions.
- the cell is a host cell.
- the cell is a dendritic cell (DC).
- the present disclosure provides a method of preparing a vaccine, comprising preparing any one of the above compositions or the above described pharmaceutical composition.
- the present disclosure provides a method of generating an immune response against an alphavirus target antigen in a subject, comprising: administering to the subject any one of the above compositions or the above described pharmaceutical composition.
- the subject has not been infected with an alphavirus.
- the alphavirus target antigen is from an alphavirus, wherein the alphavirus comprises Chikungunya virus (CHIKV), o'nyong-nyong virus (ONNV), Ross River virus (RRV), Mayaro fever virus (MAYV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalomyelitis virus (WEEV), Eastern equine encephalitis virus (EEEV), or any combination thereof.
- CHIKV Chikungunya virus
- ONNV o'nyong-nyong virus
- RRV Ross River virus
- MYV Mayaro fever virus
- VEEV Venezuelan equine encephalitis virus
- WEEV Western equine encephalomyelitis virus
- EEEV Eastern equine encephalitis virus
- the present disclosure provides a method of preventing a Chikungunya virus infection in a subject, the method comprising administering to the subject a composition comprising: a replication defective virus vector comprising a deletion in an E2b gene region; and a nucleic acid sequence encoding at least one Chikungunya target antigen.
- the subject has preexisting immunity to an adenovirus or an adenovirus vector.
- the subject is a human or a non-human animal.
- the administering is intravenously, subcutaneous ly, intralymphatically, intratumorally, intradermally, intramuscularly, intraperitoneally, intrarectally, intravaginally, intranasally, orally, via bladder instillation, or via scarification.
- FIG. 1 exemplifies one structure of an alphavirus genome.
- FIG. 2 exemplifies Ad5 [E1-, E2b-]-CHIKV str vaccine derived from nucleotide positions 7567 to 1 1313 of SEQ ID NO: 1 that has been generated.
- FIG. 3 exemplifies CHIKV structural protein expression in HEK 293 infected with Ad5 [E 1-, E2b-]-CHIKV str vaccine, or mock infected. CHIKV protein expression was detected by western blot.
- FIG. 4 exemplifies results of a challenge study performed using Ad5 [E1 -, E2b-]-CHIKV str vaccine. Mice were vaccinated 2 times at days -14 and -7 and then challenged on day 0 with a lethal amount of CHIKV injected into the footpad.
- FIG. 4A exemplifies mice vaccinated with Ad5 [E 1-, E2b-]-CHIKV str vaccine survive CHIKV infection.
- FIG. 4B exemplifies mice vaccinated with Ad5 [E 1-, E2b-]-CHIKV S i r vaccine maintain their pre-challenge weight.
- FIG. 4C exemplifies mice vaccinated with Ad5 [E1-, E2b-]-CHIKV str vaccine resolved
- FIG. 5B illustrates the translation products of the gene construct in FIG. 5A and the stoichiometric abundance of each product.
- FIG. 6 illustrates cell-mediated immune (CMI) responses (IFN- ⁇ ) and cytolytic T lymphocyte (CTL) responses and Granzyme B responses in splenocytes from immunized or control mice using an ELISpot assay.
- C57BL/6 mice were immunized two times at two-week intervals with lxlO 10 virus particles (VPs) Ad5 [E 1-, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with lxlO 10 VPs Ad5 [E1 -, E2b-]-null (empty vector control).
- VPs virus particles
- splenocytes from individual mice were tested for induction of immune responses after exposure of cells to Chikungunya virus peptides.
- the data show the cumulative number of spot forming cells (SFCs) per 10 6 splenocytes after exposure to three separate pools of Chikungunya virus peptides (peptide numbers for Chikungunya were large enough to merit division into three separate pools to use in assays - CHIKV peptide pool 1 comprised peptides 1- 103, CHIKV peptide pool 2 comprising peptides 104-207, and peptide pool 3 comprising peptides 208-310). Additional splenocytes were separately exposed to an SI V-nef peptide pools as a negative control prior to assay measurements.
- FIG. 6A illustrates CMI responses in Ad5 [E1-, E2b-]-CHIK immunized and control C57BL/6 mice as measured by IFN- ⁇ secreting SFCs using an ELISpot assay.
- FIG. 6B illustrates CTL responses in Ad5 [E1-, E2b-]-CHIK immunized and control C57BL/6 mice as measured by Granzyme B (GR-B) secreting SFCs using an ELISpot assay.
- GR-B Granzyme B
- FIG. 7 illustrates lymphocyte activation in splenocytes from immunized or control C57BL/6 mice as measured by intracellular expression of IFN- ⁇ or IFN-y/TNF-a analyzed by flow cytometry.
- C57BL/6 mice were immunized two times at two-week intervals with lxlO 10 VPs Ad5 [E1-, E2b-]- CHIK vaccine comprising SEQ ID NO: 1 or with l xlO 10 VPs Ad5 [E 1-, E2b-]-null (empty vector controls).
- splenocytes from individual mice were exposed to three separate pools of Chikungunya virus peptides (peptide numbers for Chikungunya were large enough to merit division into three separate pools to use in assays - CHIKV peptide pool 1 comprised peptides 1 - 103, CHIKV peptide pool 2 comprising peptides 104-207, and peptide pool 3 comprising peptides 208-310) and analyzed by flow cytometry for induction of intracellular cytokine expression.
- FIG. 7A illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN- ⁇ in CD8+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools).
- FIG. 7B illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN- ⁇ in CD4+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools).
- FIG. 7C illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN- ⁇ and TNF-a in CD8+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools).
- FIG. 8 illustrates anti-Chikungunya envelope protein-2 antibody responses in immunized mice as compared to control mice as measured by a quantitative enzyme-linked immunosorbent assay (ELISA).
- C57BL/6 mice were immunized two times at two-week intervals with lxl O 10 VPs Ad5 [E 1 -, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with l xl O 10 VPs Ad5 [E1 -, E2b-]-null (empty vector control).
- lxl O 10 VPs Ad5 [E 1 -, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with l xl O 10 VPs Ad5 [E1 -, E2b-]-null (empty vector control).
- lxl O 10 VPs Ad5 [E 1 -, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with l xl O 10
- alphavirus antigens such as capsid, E3 A LPHA, E2ALPHA, 6K, and E I ALPHA and nonstructural proteins such as, nsP l , nsP2, nsP3, and nsP4 can be used, for example, in a vaccine composition or a composition comprising an adenovirus vector.
- Non-structural alphavirus antigens can also be used in certain aspects. Studies have shown that the non-structural proteins involved in replication of the genome contain conserved regions that can provide a wider range of alphavirus protection when used in experimental vaccines, including those employing Ad5 vectors.
- ranges such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
- ranges include the range endpoints.
- adenovirus can refer to a group of non-enveloped DNA viruses from the family Adenoviridae. In addition to human hosts, these viruses can be found in, but are not limited to, avian, bovine, porcine and canine species.
- the use of any adenovirus from any of the four genera of the family Adenoviridae e.g., Aviadenovirus, Mastadenovirus, Atadenovirus and Siadenovirus
- Ad also pertains to genetic derivatives of any of these viral serotypes, including but not limited to, genetic mutation, deletion or transposition of homologous or heterologous DNA sequences.
- a "helper adenovirus” or “helper virus” can refer to an Ad that can supply viral functions that a particular host cell cannot (the host can provide Ad gene products such as E l proteins).
- This virus can be used to supply, in trans, functions (e.g., proteins) that are lacking in a second virus, or helper dependent virus (e.g., a gutted or gutless virus, or a virus deleted for a particular region such as E2b or other region as described herein); the first replication- incompetent virus can be said to "help" the second, helper dependent virus thereby permitting the production of the second viral genome in a cell.
- Ad5 null can refer to a non-replicating Ad that does not contain any heterologous nucleic acid sequences for expression.
- First Generation adenovirus can refer to an Ad that has the early region 1 (E l ) deleted. In additional cases, the nonessential early region 3 (E3) can also be deleted.
- gutted or "gutless,” as used herein, can refer to an adenovirus vector that has been deleted of all viral coding regions.
- transfection can refer to the introduction of foreign nucleic acid into eukaryotic cells. Transfection can be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
- stable transfection or “stably transfected” can refer to the introduction and integration of foreign nucleic acid, DNA or RNA, into the genome of the transfected cell.
- stable transfectant can refer to a cell which has stably integrated foreign DNA into the genomic DNA.
- reporter gene can indicate a nucleotide sequence that encodes a reporter molecule (including an enzyme).
- a "reporter molecule” can be detectable in any of a variety of detection systems, including, but not limited to enzyme-based detection assays (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems.
- the E. coli ⁇ -galactosidase gene (available from Pharmacia Biotech, Pistacataway, N.J.), green fluorescent protein (GFP) (commercially available from Clontech, Palo Alto, Calif), the human placental alkaline phosphatase gene, the chloramphenicol acety transferase (CAT) gene or other reporter genes that are known to the art can be employed.
- GFP green fluorescent protein
- CAT chloramphenicol acety transferase
- heterologous nucleic acid sequence can refer to a nucleotide sequence that is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature.
- Heterologous nucleic acid can include a nucleotide sequence that is naturally found in the cell into which it is introduced or the heterologous nucleic acid can contain some modification relative to the naturally occurring sequence.
- transgene can refer to any gene coding region, either natural or heterologous nucleic acid sequences or fused homologous or heterologous nucleic acid sequences, introduced into the cells or genome of a test subject.
- transgenes are carried on any viral vector that is used to introduce the transgenes to the cells of the subject.
- the term "Second Generation Adenovirus,” as used herein, can refer to an Ad that has all or parts of the E l, E2, E3, and, in certain embodiments, E4 DNA gene sequences deleted (removed) from the virus.
- compositions and methods for producing a vaccine that generates immune responses against various alphaviruses using an adenovirus vector that allows for vaccinations to generate broadly reactive immune responses against alphaviruses.
- the target antigens are comprised of antigens derived from various alphavirus proteins.
- the alphavirus proteins can be derived from any alphavirus, including but not limited to Chikungunya virus (CHIKV), o'nyong-nyong virus (ONNV), Ross River virus (RRV), Mayaro fever virus (MAYV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalomyelitis virus (WEEV), and Eastern equine encephalitis virus (EEEV).
- the envelope glycoproteins E l ALPHA and E2ALPHA can form heterodimers that associate to form trimeric spikes on surface of the virion.
- the alphavirus replicative cycle can begin when the trimeric spikes bind host-cell receptors and can cause the endocytosis of virions.
- the low pH of the endosomes can induce fusion of the viral and endosomal membranes thereby releasing the viral genome into the cytosol of the cell.
- the genomic R A of alphaviruses can serve as an mRNA which, like cellular mRNAs, can be capped with 7-methylguanosine and can be polyadenylated.
- the first approximately 7 kB of the genome can encode the non-structural proteins that comprise the viral replicase and transcriptase.
- the final approximately 5 kB of the genome can encode the structural proteins.
- the viral replicase proteins, nsP l , nsP2, nsP3, and nsP4 produce anti-genome which then can serve as a template for production of genome and two mRNAs, one for the non-structural proteins and one for the structural proteins.
- the structural proteins can be translated as a polyprotein that can be processed by a combination of a serine protease activity on the capsid protein and cellular enzymes in the secretory system (e.g., signal peptidase and furin).
- the envelope glycoproteins, E I ALPHA and E2 A LPHA can transit from the secretory system to the plasma membrane where they can be found in the fully processed, mature form.
- E l ALPHA and ⁇ 2 ALPHA recruit nucleocapsids (capsid protein shells with genomic RNA inside), and virions can be formed by budding from the plasma membrane.
- Target antigens can include proteins, or variants or fragments thereof, associated with alphaviruses, such as C, E3 ALPHA, E2 A LPHA, 6K, E I ALPHA, nsP l , nsP2, nsP3, and nsP4.
- the nucleic acid sequence in the composition described herein comprises the CHIKV gene (nucleotides 7567-1 1313 of SEQ ID NO: 1) with a gene ID 956308, which has a gene symbol CHIKVgp2 and encodes a polyprotein containing C, E3, E2, 6K, and E l proteins.
- the nucleic acid sequence in the composition described herein comprises the CHIKV gene (nucleotides 77-7501 of SEQ ID NO: 1) with a gene ID 953609 which has a gene symbol CHIKVgpl (encoding a polyprotein containing nsp l , nsp2, nsp3, and nsp4 proteins).
- the nucleic acid sequence in the composition described herein comprises a nucleic acid sequence encoding a Chikungunya virus structural polyprotein (e.g., NP_690589) or fragments thereof. In some embodiments, the nucleic acid sequence in the composition described herein comprises a nucleic acid sequence encoding a Chikungunya virus nonstructural polyprotein (e.g., NP_690588) or fragments thereof.
- the at least one target antigen is a CHIKV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 1.
- the at least one target antigen is a CHIKV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, or any combination thereof.
- the at least one target antigen is an ONNV antigen encoded by the nucleotide sequence set forth in SEQ ID NO: 4.
- the at least one target antigen used herein is an ONNV structural antigen having an amino acid sequence set forth in SEQ ID NO: 5, an ONNV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 6, or a combination thereof.
- the at least one target antigen is an ONNV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 4.
- the at least one target antigen is a ONNV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, or any combination thereof.
- the at least one target antigen is a MAYV antigen that is encoded by a sequence set forth in SEQ ID NO: 7.
- the at least one target antigen is a MAYV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9, or any combination thereof.
- the at least one target antigen is a RRV antigen that is encoded by a sequence set forth in SEQ ID NO: 10.
- the at least one target antigen used herein is a RRV structural antigen having an amino acid sequence set forth in SEQ ID NO: 1 1 , a RRV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 12, or a combination thereof.
- the at least one target antigen is a RRV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 10.
- the at least one target antigen is a RRV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 1 1 , SEQ ID NO: 12, or any combination thereof.
- the at least one target antigen is a VEEV antigen that is encoded by a sequence set forth in SEQ ID NO: 13.
- the at least one target antigen used herein is an EEEV structural antigen having an amino acid sequence set forth in SEQ ID NO: 17, an EEEV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 18, or a combination thereof.
- the at least one target antigen is a EEEV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 16.
- the at least one target antigen is a EEEV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, or any combination thereof.
- the at least one target antigen is a WEEV antigen that is encoded by a sequence set forth in SEQ ID NO: 19.
- the at least one target antigen used herein is a WEEV structural antigen having an amino acid sequence set forth in SEQ ID NO: 20, a WEEV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 21, or a combination thereof.
- the at least one target antigen is a WEEV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 19.
- the at least one target antigen is a WEEV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 20, SEQ ID NO: 21 , or any combination thereof.
- adenovirus vectors can be used in compositions and methods for the delivery of alphavirus antigens.
- the vector can be an expanded gene-carrying/cloning capacity of up to 12 kb, compared to the 7 kb capacity of current Ad5 [E 1-] vectors, which can be sufficient to allow inclusion of multiple genes (Amalfitano A et al. J Virol. 1998 72:926-33; Seregin SS and Amalfitano A Expert Opin Biol Ther. 2009 9: 1521 -31). Additional deletions of the E2b region can confer advantageous immune properties such as eliciting potent immune responses to specific antigens while minimizing immune responses to Ad5 viral proteins.
- Ad5 vector platforms can give the opportunity to develop a novel broadly cross-reactive vaccine for alphavirus.
- This vector can be delivered directly by subcutaneous injection for exposure of defined alphavirus antigens to antigen- presenting cells (APCs) that induce potent immune responses.
- APCs antigen- presenting cells
- the Ad5 recombinant vector can replicate episomally and may not insert the genome into the host cell genome, thereby ensuring that there is no gene integration or disruption of vital cellular gene functions (Imler JL Vaccine. 1995 13: 1 143-51 ; Ertl HC, Xiang Z J Immunol. 1996 156:3579-82; Amalfitano, A Curr Opin Mol Ther. 2003 5:362-6).
- Ad5 [E 1-, E2b-] vectors can display reduced inflammation during the first 24 to 72 hours after injection compared to current Ad5 [E 1 -] vectors (Nazir SA, Metcalf JP J Investig Med. 2005 53:292-304; Schaack J Proc Natl Acad Sci U S A. 2004 101 :3124-9; Schaack J Viral Immunol. 2005 18:79-88).
- the lack of Ad5 [E 1 -, E2b-] late gene expression can render infected cells less vulnerable to anti-Ad5 activity and can permit them to produce and express the transgene for extended periods of time (Gabitzsch ES, Jones FR J Clin Cell Immunol. 201 1 S4.001.
- deletion of the E2b region can confer advantageous immune properties, such as eliciting potent immune responses to specific antigens, while minimizing immune responses to Ad5 proteins even in the presence of pre-existing Ad5 immunity.
- Results demonstrated the ability of recombinant Ad5 [E1-, E2b-] platform-based vaccines to overcome pre-existing and/or Ad5 vector-induced immunity and induce significant protective immune responses.
- E2b deleted adenovirus vectors such as those described in U.S. Patent Nos. 6,063,622; 6,451 ,596; 6,057, 158: and 6,083,750 (all incorporated herein in their entirety by reference).
- adenovirus vectors containing deletions in the E2b region can be provided in certain aspects. Propagation of these E2b deleted adenovirus vectors requires cell lines that can express the deleted E2b gene products.
- packaging cell lines for example E.C7 (formally called C-7), derived from the HEK-203 cell line (Amalfitano A et al. Proc Natl Acad Sci USA 1996 93:3352-56; Amalfitano A et al. Gene Ther 1997 4:258-63).
- the E2b gene products, DNA polymerase and preterminal protein can be constitutively expressed in E.C7, or similar cells along with the El gene products.
- Transfer of gene segments from the Ad genome to the production cell line can have immediate benefits: (1) increased carrying capacity of the recombinant DNA polymerase and preterminal protein-deleted adenovirus vector, since the combined coding sequences of the DNA polymerase and preterminal proteins that can be theoretically deleted approaches 4.6 kb; and, (2) a decreased potential of RCA generation, since two or more independent recombination events would be required to generate RCA.
- genes critical to the viral life cycle are deleted (e.g., the E2b genes)
- a further crippling of Ad to replicate or express other viral gene proteins can occur. This can decrease immune recognition of virally infected cells, and can allow for extended durations of foreign transgene expression.
- E l DNA polymerase, and preterminal protein deleted vectors, however, include their inability to express the respective proteins from the E l and E2b regions, as well as a predicted lack of expression of most of the viral structural proteins.
- MLP major late promoter
- the major late promoter (MLP) of Ad is responsible for transcription of the late structural proteins LI through L5 (Doerfler, In Adenovirus DNA, The Viral Genome and Its Expression (Martinus Nijhoff Publishing Boston, 1986)).
- the MLP is minimally active prior to Ad genome replication, the highly toxic Ad late genes are primarily transcribed and translated from the MLP only after viral genome replication has occurred (Thomas and Mathews Cell 1980 22:523).
- This cis-dependent activation of late gene transcription is a feature of DNA viruses in general, such as in the growth of polyoma and SV-40.
- the DNA polymerase and preterminal proteins are absolutely required for Ad replication (unlike the E4 or protein IX proteins) and thus their deletion is extremely detrimental to adenovirus vector late gene expression, and the toxic effects of that expression in cells such as APCs.
- the adenovirus vectors contemplated for use include E2b deleted adenovirus vectors that have a deletion in the E2b region of the Ad genome and the E l region but do not have any other regions of the Ad genome deleted.
- the adenovirus vectors contemplated for use can include E2b deleted adenovirus vectors that have a deletion in the E2b region of the Ad genome and deletions in the E l and E3 regions, but no other regions deleted.
- the adenovirus vectors contemplated for use can include adenovirus vectors that have a deletion in the E2b region of the Ad genome and deletions in the E l , E3 and partial or complete removal of the E4 regions but no other deletions.
- the adenovirus vectors contemplated for use include adenovirus vectors that have a deletion in the E2b region of the Ad genome and deletions in the E l and E4 regions but no other deletions.
- the adenovirus vectors contemplated for use can include adenovirus vectors that have a deletion in the E2a, E2b, and E4 regions of the Ad genome but no other deletions.
- the adenovirus vectors for use herein comprise vectors having the El and DNA polymerase functions of the E2b region deleted but no other deletions.
- the adenovirus vectors for use herein have the El and the preterminal protein functions of the E2b region deleted and no other deletions.
- the adenovirus vectors for use herein have the El , DNA polymerase and the preterminal protein functions deleted, and no other deletions.
- the adenovirus vectors contemplated for use herein are deleted for at least a portion of the E2b region and the El region, but are not "gutted" adenovirus vectors.
- the vectors can be deleted for both the DNA polymerase and the preterminal protein functions of the E2b region.
- the deletion is of more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within the E2b region of the Ad genome.
- An E2b deletion can be a deletion that prevents expression and/or function of at least one E2b gene product and therefore, can encompass deletions within exons encoding portions of E2b-specific proteins as well as deletions within promoter and leader sequences.
- an E2b deletion is a deletion that prevents expression and/or function of one or both of the DNA polymerase and the preterminal protein of the E2b region.
- E2b deleted can refer to one or more point mutations in the DNA sequence of this region of an Ad genome such that one or more encoded proteins is non-functional. Such mutations can include residues that are replaced with a different residue leading to a change in the amino acid sequence that result in a nonfunctional protein.
- regions of the Ad genome can be deleted.
- regions of the Ad genome can refer to a specific DNA sequence that is mutated in such a way so as to prevent expression and/or function of at least one gene product encoded by that region.
- to be “deleted” in a particular region can refer to a specific DNA sequence that is deleted (removed) from the Ad genome in such a way so as to prevent the expression and/or the function encoded by that region (e.g., E2b functions of DNA polymerase or preterminal protein function).
- deleted or "containing a deletion" within a particular region can refer to a deletion of at least one base pair within that region of the Ad genome.
- more than one base pair is deleted and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, or 150 base pairs are deleted from a particular region.
- the deletion is more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within a particular region of the Ad genome.
- any one of the above described deletions can also be a result of translocation of two or more base pairs.
- deletions are such that expression and/or function of the gene product encoded by the region can be prevented.
- deletions can encompass deletions within exons encoding portions of proteins as well as deletions within promoter and leader sequences.
- "deleted" in a particular region of the Ad genome can refer to one or more point mutations in the DNA sequence of this region of an Ad genome such that one or more encoded proteins is nonfunctional. Such mutations can include residues that are replaced with a different residue leading to a change in the amino acid sequence that result in a nonfunctional protein.
- the adenovirus vectors comprising one or more deletions can be generated using recombinant techniques known in the art (see e.g., Amalfitano et al. J. Virol. 1998 72:926-33; Hodges, et al., J Gene Med 2000 2:250-59).
- the adenovirus vectors for use can be successfully grown to high titers using an appropriate packaging cell line that constitutively expresses E2b gene products and products of any of the necessary genes that can have been deleted.
- HEK-293-derived cells that not only constitutively express the El and DNA polymerase proteins, but also the Ad-preterminal protein, can be used.
- E.C7 cells are used to successfully grow high titer stocks of the adenovirus vectors (see e.g., Amalfitano et al. J. Virol. 1998 72:926-33; Hodges et al. J Gene Med 2000 2:250-59)
- the proteins encoded by the targeted genes have to first be coexpressed in HEK-293 cells, or similar, along with the El proteins. Therefore, only those proteins which are non-toxic when coexpressed constitutively (or toxic proteins inducibly- expressed) can be utilized.
- Coexpression in HEK-293 cells of the El and E4 genes has been demonstrated (utilizing inducible, not constitutive, promoters) (Yeh et al. J. Virol. 1996 70:559; Wang et al. Gene Therapy 1995 2:775; and Gorziglia et al. J. Virol. 1996 70:4173).
- the El and protein IX genes (a virion structural protein) have been coexpressed (Caravokyri and Leppard J. Virol. 1995 69:6627), and coexpression of the El , E4, and protein IX genes has also been described (Krougliak and Graham Hum. Gene Ther. 1995 6: 1575).
- the El and 100k genes have been successfully expressed in transcomplementing cell lines, as have El and protease genes (Oualikene et al. Hum Gene Ther 2000 1 1 : 1341-53; Hodges et al. J. Virol 2001 75:5913-20).
- cell lines that have high-level, constitutive coexpression of the El , DNA polymerase, and preterminal proteins, without toxicity can be desirable for use in propagating Ad for use in multiple vaccinations.
- These cell lines can permit the propagation of adenovirus vectors deleted for the El, DNA polymerase, and preterminal proteins.
- the recombinant Ad can be propagated using techniques known in the art. For example, in certain embodiments, tissue culture plates containing E.C7 cells are infected with the adenovirus vector virus stocks at an appropriate MOI (e.g., 5) and incubated at 37.0°C for 40-96 h. The infected cells can be harvested, resuspended in 10 mM Tris-CI (pH 8.0), and sonicated, and the virus can be purified by two rounds of cesium chloride density centrifugation.
- MOI e.g. 5
- 10 mM Tris-CI pH 8.0
- the virus containing band is desalted over a Sephadex CL-6B column (Pharmacia Biotech, Piscataway, N.J.), sucrose or glycerol is added, and aliquots are stored at -80°C.
- the virus can be placed in a solution designed to enhance its stability, such as A 195 (Evans et al. J Pharm Sci 2004 93:2458-75). The titer of the stock can be measured (e.g., by measurement of the optical density at 260 nm of an aliquot of the virus after SDS lysis).
- plasmid DNA either linear or circular, encompassing the entire recombinant E2b deleted adenovirus vector can be transfected into E.C7, or similar cells, and can be incubated at 37.0°C until evidence of viral production is present (e.g., the cytopathic effect). The conditioned media from these cells can then be used to infect more E.C7, or similar cells, to expand the amount of virus produced, before purification.
- Purification can be accomplished by two rounds of cesium chloride density centrifugation or selective filtration.
- the virus can be purified by column chromatography, using commercially available products (e.g., Adenopure from Puresyn, Inc., Malvern, PA) or custom made chromatographic columns.
- the recombinant Ad can comprise enough of the virus to ensure that the cells to be infected are confronted with a certain number of viruses.
- a stock of recombinant Ad particularly, an RCA-free stock of recombinant Ad.
- the preparation and analysis of Ad stocks is well known in the art. Viral stocks can vary considerably in titer, depending largely on viral genotype and the protocol and cell lines used to prepare them.
- the viral stocks can have a titer of at least about 10 6 , 10 7 , or 10 8 virus particles (VPs)/ml, and many such stocks can have higher titers, such as at least about 10 9 , 10 10 , lO 1 1 , or 10 12 VPs/ml.
- VPs virus particles
- the adenovirus vectors described herein comprise heterologous nucleic acid sequences that encode one or more target antigens of interest such as alphavirus target antigens, fragments or fusions thereof, against which it is desired to generate an immune response.
- the adenovirus vectors comprise heterologous nucleic acid sequences that encode several proteins, fusions thereof or fragments thereof, which can modulate the immune response.
- Certain aspects provide the Second Generation E2b deleted adenovirus vectors that comprise a heterologous nucleic acid sequence such as an alphavirus target antigen.
- polynucleotides can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or can be adapted to express target antigens as described herein, fragments of antigens, peptides and the like. Such segments can be naturally isolated, or modified synthetically by the hand of man.
- Polynucleotides can comprise a native sequence (i.e., an endogenous sequence that encodes a target antigen polypeptide/protein/epitope or a portion thereof) or can comprise a sequence that encodes a variant or derivative of such a sequence.
- polynucleotide variants having substantial identity to native sequences encoding proteins (e.g., target antigens of interest) as described herein, for example those comprising at least 70% sequence identity, particularly at least 75% up to 99% or higher, sequence identity compared to a native polynucleotide sequence encoding the polypeptides using the methods described herein (e.g., BLAST analysis using standard parameters, as described below).
- proteins e.g., target antigens of interest
- sequence identity compared to a native polynucleotide sequence encoding the polypeptides using the methods described herein (e.g., BLAST analysis using standard parameters, as described below).
- polynucleotide variants can contain one or more substitutions, additions, deletions and/or insertions, particularly such that the immunogenicity of the epitope of the polypeptide encoded by the variant polynucleotide or such that the immunogenicity of the heterologous target protein may not be substantially diminished relative to a polypeptide encoded by the native polynucleotide sequence.
- the polynucleotide variants can encode a variant of the target antigen, or a fragment (e.g., an epitope) thereof wherein the propensity of the variant polypeptide or fragment (e.g., epitope) thereof to react with antigen-specific antisera and/or T-cell lines or clones may not be substantially diminished relative to the native polypeptide.
- variants can also encompass homologous genes of xenogeneic origin.
- polynucleotides that comprise or consist of at least about 5 up to a 1000 or more contiguous nucleotides encoding a polypeptide, including target protein antigens, as described herein, as well as all intermediate lengths there between.
- intermediate lengths can mean any length between the quoted values, such as 16, 17, 18, 19, etc.; 21 , 22, 23, etc.; 30, 31, 32, etc.; 50, 51 , 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151 , 152, 153, etc.; including all integers through 200-500; 500-1 ,000, and the like.
- a polynucleotide sequence as described herein can be extended at one or both ends by additional nucleotides not found in the native sequence encoding a polypeptide as described herein, such as an epitope or heterologous target protein.
- This additional sequence can consist of 1 up 20 nucleotides or more, at either end of the disclosed sequence or at both ends of the disclosed sequence.
- illustrative polynucleotide segments with total lengths of about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations.
- two sequences can be said to be “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences can be performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
- a “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
- Optimal alignment of sequences for comparison can be conducted using the Megalign program in the Lasergene suite of bio informatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff MO (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345- 358; Hein J Unified Approach to Alignment and Phylogenes, pp.
- optimal alignment of sequences for comparison can be conducted by the local identity algorithm of Smith and Waterman, Add. APL. Math 1981 2:482, by the identity alignment algorithm of Needleman and Wunsch J. Mol. Biol. 1970 48:443, by the search for similarity methods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 1988 85 :2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wl), or by inspection.
- BLAST and BLAST 2.0 are described in Altschul et al., Nucl. Acids Res. 1977 25 :3389-3402, and Altschul et al. J. Mol. Biol. 1990 215 :403-10, respectively.
- BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides.
- Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
- cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). Extension of the word hits in each direction can be halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
- the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
- the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- the percentage can be calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
- nucleotide sequences that encode a particular antigen of interest, or fragment thereof, as described herein. Some of these polynucleotides can bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in certain aspects. Further, alleles of the genes comprising the polynucleotide sequences provided herein are also contemplated. Alleles can be endogenous genes that can be altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein can, but need not, have an altered structure or function. Alleles can be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
- a mutagenesis approach such as site-specific mutagenesis, is employed for the preparation of variants and/or derivatives of the target antigen sequences, or fragments thereof, as described herein.
- site-specific mutagenesis By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them.
- nucleotide sequences encoding the polypeptide, or functional equivalents can be inserted into an appropriate Ad as described elsewhere herein using recombinant techniques known in the art.
- the appropriate adenovirus vector can contain the necessary elements for the transcription and translation of the inserted coding sequence and any desired linkers. Methods that are well known to those skilled in the art can be used to construct these adenovirus vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements.
- a variety of vector/host systems can be utilized to contain and produce polynucleotide sequences. These can include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA vectors; yeast transformed with yeast vectors; insect cell systems infected with virus vectors (e.g., baculovirus); plant cell systems transformed with virus vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA vectors
- yeast transformed with yeast vectors insect cell systems infected with virus vectors (e.g., baculovirus)
- plant cell systems transformed with virus vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
- virus vectors
- control elements or "regulatory sequences” present in an adenovirus vector can be those non-translated regions of the vector— enhancers, promoters, 5' and 3' untranslated regions— which can interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, sequences encoding a polypeptide of interest can be ligated into an Ad transcription/translation complex consisting of the late promoter and tripartite leader sequence.
- Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing the polypeptide in infected host cells (Logan J and Shenk T (1984) Proc. Natl. Acad. Sci 1984 87:3655-59).
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
- Transcriptional enhancers can comprise one element, at least two elements, at least three elements, at least four elements, at least five elements, or at least six elements.
- a variety of protocols for detecting and measuring the expression of polynucleotide-encoded products are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- FACS fluorescence activated cell sorting
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide can be used for some applications, but a competitive binding assay can also be employed.
- elements that increase the expression of the desired target antigen are incorporated into the nucleic acid sequence of the adenovirus vectors described herein.
- Such elements include internal ribosome binding sites (IRES; Wang and Siddiqui Curr. Top. Microbiol. Immunol 1995 203:99; Ehrenfeld and Semler Curr. Top. Microbiol. Immunol. 1995 203:65; Rees et al., Biotechniques 1996 20: 102; Sugimoto et al. Biotechnology 1994 2:694).
- IRES can increase translation efficiency.
- Other sequences can also enhance expression. For some genes, sequences especially at the 5 ' end can inhibit transcription and/or translation. These sequences can be palindromes that can form hairpin structures. Any such sequences in the nucleic acid to be delivered can be deleted or not deleted.
- transcript levels of the transcript or translated product can be assayed to confirm or ascertain which sequences affect expression.
- Transcript levels can be assayed by any known method, including Northern blot hybridization, Rnase probe protection and the like.
- Protein levels can be assayed by any known method, including ELISA.
- the adenovirus vectors comprising heterologous nucleic acid sequences can be generated using recombinant techniques known in the art, such as those described in Maione et al. Proc Natl Acad Sci USA 2001 98:5986-91 ; Maione et al. Hum Gene Ther 2000 1 :859-68; Sandig et al.
- the adenovirus vectors can comprise nucleic acid sequences that can encode several alphavirus target proteins or antigens of interest.
- the vectors can contain nucleic acid encoding 1 to 4 or more different target antigens of interest.
- the target antigens can be a full length protein or can be a fragment (e.g., an epitope) thereof.
- the adenovirus vectors can contain nucleic acid sequences encoding multiple fragments or epitopes from one target protein of interest or can contain one or more fragments or epitopes from numerous different target alphavirus antigen proteins of interest.
- the nucleic acid sequences encode a plurality of alphavirus target antigens.
- the nucleic acid sequence encoding the plurality of alphavirus target antigens can comprise a plurality of gene inserts each corresponding to a target antigen and wherein each gene insert is separated by a nucleic acid sequence encoding a self-cleaving 2A peptide.
- the self- cleaving 2A peptide i.e., the cleavable linker
- the self- cleaving 2A peptide is derived from Porcine teschovirus- 1 or Thosea asigna virus or the like.
- cleavable linkers can include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof and combinations thereof.
- the linkers include the picornaviral 2A-like linker, CHYSEL sequences of Porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof.
- the adenovirus vector can also include nucleic acid sequences that encode proteins that increase the immunogenicity of the target antigen.
- the protein produced following immunization with the adenovirus vector containing such a protein can be a fusion protein comprising the target antigen of interest fused to a protein that increases the immunogenicity of the target antigen of interest.
- Certain embodiments provide a combination immunotherapy and vaccine composition for the treatment and prevention infectious diseases. Some embodiments provide combination multi-targeted vaccines, immunotherapies, and methods for enhanced therapeutic response to complex diseases such as infectious diseases. Each component of the combination therapy can be independently included in a vaccine composition for prevention of Chikungunya infection or infection by any alphavirus.
- Treatment can refer to administration of a therapeutically effective dose of a vaccine of this disclosure to a subject.
- the treatment can be administered in a pharmaceutical composition to a subject.
- the subject can also be healthy and disease free at the time of treatment and, in this case, the treatment can be referred to as a preventative vaccination.
- the subject can be suffering from a disease condition at the time of treatment and, in this case, the treatment can be referred to as therapeutic vaccination.
- a “subject” can refer to any animal, including, but not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats and fowls.
- a “subject” can be used herein interchangeably with “individual” or "patient.”
- the vector comprises at least one antigen. In some aspects, the vector comprises at least two antigens. In some aspects, the vaccine formulation comprises 1 : 1 ratio of vector to antigen. In some aspects, the vaccine comprises 1 :2 ratio of vector to antigen. In some aspects, the vaccine comprises 1 :3 ratio of vector to antigen. In some aspects, the vaccine comprises 1 :4 ratio of vector to antigen. In some aspects, the vaccine comprises 1 :5 ratio of vector to antigen. In some aspects, the vaccine comprises 1 :6 ratio of vector to antigen. In some aspects, the vaccine comprises 1 :7 ratio of vector to antigen. In some aspects, the vaccine comprises 1 :8 ratio of vector to antigen. In some aspects, the vaccine comprises 1 :9 ratio of vector to antigen. In some aspects, the vaccine comprises 1 : 10 ratio of vector to antigen.
- the vaccine is a combination vaccine, wherein the vaccine comprises at least two vectors each containing at least a single antigen.
- composition that comprises multiple antigens can be present at various ratios.
- formulations with more than one vector can have various ratios.
- immunotherapies or vaccines can have two different vectors in a stoichiometry of 1 : 1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 15, 1 :20, 1 :30, 2: 1 , 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3 : 1 , 3 :3, 3 :4, 3 :5, 3:6, 3:7, 3 :8, 3 : 1 , 3:3, 3 :4, 3 :5, 3:6, 3:7, 3 :8, 3 : 1 , 3:3, 3 :4, 3 :5, 3 :6, 3 :7, 3 :8, 4: 1 , 4:3, 4:5, 4:6, 4:7, 4:8, 5 : 1 , 5 :
- At least one of the recombinant nucleic acid vectors is a replication defective virus vector that comprises a replication defective adenovirus 5 vector comprising a first identity value.
- the replication defective adenovirus vector comprises a deletion in the E2b gene region.
- the replication defective adenovirus vector further comprises a deletion in the E l gene region.
- the replication defective adenovirus vector comprises a deletion in an E l gene region, and E2b gene region, an E3 gene region, an E4 gene region, or any combination thereof.
- co-stimulatory molecules can be incorporated into said the vaccine to increase immunogenicity.
- Initiation of an immune response can require at least two signals for the activation of I T cells by APCs (Damle, et al. J Immunol 148: 1985-92 ( 1992); Guinan, et al. Blood 84:3261 -82 ( 1994); Hellstrom, et al. Cancer Chemother Pharmacol 38:S40-44 ( 1996); Hodge, et al. Cancer Res 39:5800-07 ( 1999).
- An antigen specific first signal can be delivered through the T cell receptor (TCR) via the peptide/major histocompatibility complex (MHC) and can cause the T cell to enter the cell cycle.
- a second, or costimulatory, signal can be delivered for cytokine production and proliferation.
- At least three distinct molecules normally found on the surface of professional antigen presenting cells can be capable of providing the second signal critical for T cell activation: B7- 1 (CD80), ICAM-1 (CD54), and LFA-3 (human CD58) (Damle, et al. J Immunol 148 : 1985-92 (1992) ; Guinan, et al. Blood 84 : 3261-82 (1994) ; Wingren, et al. Crit Rev Immunol 15 : 235-53 (1995) ; Parra, et al. Scand. J Immunol 38 : 508-14 (1993) ; Hellstrom, et al.
- costimulatory molecules can have distinct T cell ligands.
- B7-1 can interact with the CD28 and CTLA-4 molecules
- ICAM-1 can interact with the CDl la/CD18 (LFA- ⁇ eta-2 integrin) complex
- LFA-3 can interact with the CD2 (LFA-2) molecules.
- a recombinant adenovirus vector that contains B7-1 , ICAM-1 , and LFA-3, respectively, that, when combined with a recombinant adenovirus-based vector vaccine containing one or more nucleic acids encoding target antigens such as alphavirus antigens, can further increase/enhance anti-alphavirus immune responses directed to specific target antigens.
- the viral vector or composition described herein can further comprise nucleic acid sequences that encode proteins, or an "immunological fusion partner," that can increase the immunogenicity of the target antigen such as Chikungunya virus antigens, or any target antigen of the present disclosure.
- the protein produced following immunization with the viral vector containing such a protein can be a fusion protein comprising the target antigen of interest fused to a protein that increases the immunogenicity of the target antigen of interest.
- combination therapy with Ad5[E l-, E2b-] vectors encoding for a Chikungunya virus antigen and an immunological fusion partner can result in boosting the immune response, such that the combination of both therapeutic moieties acts to synergistically boost the immune response more than either the Ad5[E l -, E2b-] vector encoding for a Chikungunya virus antigen alone, or the immunological fusion partner alone.
- combination therapy with an Ad5[El -, E2b-] vector encoding for a Chikungunya virus antigen and an immunological fusion partner can result in synergistic enhancement of stimulation of antigen-specific effector CD4+ and CD8+ T cells, stimulation of NK cell response directed towards killing infected cells, stimulation of neutrophils or monocyte cell responses directed towards killing infected cells via antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) mechanisms, or any combination thereof.
- ADCC antibody dependent cell-mediated cytotoxicity
- ADCP antibody dependent cellular phagocytosis
- combination therapy with an Ad5[El -, E2b-] vector encoding for a Chikungunya virus antigen and an immunological fusion partner can result in generating an immune response comprises an increase in target antigen-specific CTL activity of about 1.5 to 20, or more fold in a subject administered the adenovirus vectors as compared to a control.
- generating an immune response comprises an increase in target-specific CTL activity of about 1.5 to 20, or more fold in a subject administered the Ad5[E l -, E2b-] vector encoding for a Chikungunya virus antigen and an immunological fusion partner as compared to a control.
- generating an immune response that comprises an increase in target antigen-specific cell-mediated immunity activity as measured by ELISpot assays measuring cytokine secretion, such as interferon-gamrna (IFN- ⁇ ), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-a), or other cytokines, of about 1.5 to 20, or more fold as compared to a control.
- generating an immune response comprises an increase in target-specific antibody production of between 1.5 and 5 fold in a subject administered the Ad5[El -, E2b-] vectors encoding for a Chikungunya virus antigen and an immunological fusion partner as described herein as compared to an appropriate control.
- generating an immune response comprises an increase in target-specific antibody production of about 1.5 to 20, or more fold in a subject administered the adenovirus vector as compared to a control.
- combination therapy with an Ad5[E l-, E2b-] vector encoding for target epitope antigens and an immunological fusion partner can result in synergistic enhancement of stimulation of antigen-specific effector CD4+ and CD8+ T cells, stimulation of NK cell response directed towards killing infected cells, stimulation of neutrophils or monocyte cell responses directed towards killing infected cells via antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) mechanisms, or any combination thereof.
- ADCC antibody dependent cell-mediated cytotoxicity
- ADCP antibody dependent cellular phagocytosis
- combination therapy with Ad5[El-, E2b-] vectors encoding for a target epitope antigen and an immunological fusion partner can result in generating an immune response comprises an increase in target antigen-specific CTL activity of about 1.5 to 20, or more fold in a subject administered the adenovirus vectors as compared to a control.
- generating an immune response comprises an increase in target-specific CTL activity of about 1.5 to 20, or more fold in a subject administered the Ad5[El -, E2b-] vector encoding for target epitope antigen and an immunological fusion partner as compared to a control.
- generating an immune response that comprises an increase in target antigen-specific cell-mediated immunity activity as measured by ELISpot assays measuring cytokine secretion, such as interferon-gamma (IFN- ⁇ ), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-a), or other cytokines, of about 1.5 to 20, or more fold as compared to a control.
- cytokine secretion such as interferon-gamma (IFN- ⁇ ), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-a), or other cytokines
- generating an immune response comprises an increase in target-specific antibody production of between 1.5 and 5 fold in a subject administered the adenovirus vectors as described herein as compared to an appropriate control.
- generating an immune response comprises an increase in target- specific antibody production of about 1.5 to 20, or more fold in a subject administered the adenovirus vector as compared to a control.
- such an immunological fusion partner is derived from a Mycobacterium sp. , such as a Mycobacterium tuberculosis-derived Ral2 fragment.
- the immunological fusion partner derived from Mycobacterium sp. can be any one of the sequences set forth in SEQ ID NO: 22 - SEQ ID NO: 30 and SEQ ID NO: 93 - SEQ ID NO: 98. Oligonucleotides, Met-His tags, and enterokinase recognition sites, which can be used to construct these Mycobacterium sp. -derived Ral 2 sequences are set forth in any one of SEQ ID NO: 99 - SEQ ID NO: 106 as shown in TABLE 2.
- Ral2 compositions and methods for their use in enhancing the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences are described in U.S. Patent No. 7,009,042, which is herein incorporated by reference in its entirety.
- Ral 2 refers to a polynucleotide region that is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid.
- MTB32A is a serine protease of 32 kDa encoded by a gene in virulent and avirulent strains of M. tuberculosis.
- MTB32A The nucleotide sequence and amino acid sequence of MTB32A have been described (see, e.g., U.S. Patent No. 7,009,042; Skeiky et al., Infection and Immun. 67:3998-4007 (1999), incorporated herein by reference in their entirety).
- C-terminal fragments of the MTB32A coding sequence can be expressed at high levels and remain as soluble polypeptides throughout the purification process.
- Ral 2 can enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused.
- a Ral2 fusion polypeptide can comprise a 14 kDa C-teirninal fragment corresponding to amino acid residues 192 to 323 of MTB32A.
- Ral 2 polynucleotides generally can comprise at least about 15, 30, 60, 100, 200, 300, or more nucleotides that encode a portion of a Ral2 polypeptide.
- Ral2 polynucleotides can comprise a native sequence (i.e., an endogenous sequence that encodes a Ral2 polypeptide or a portion thereof) or can comprise a variant of such a sequence.
- Ral2 polynucleotide variants can contain one or more substitutions, additions, deletions and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished, relative to a fusion polypeptide comprising a native Ral2 polypeptide.
- Variants can have at least about 70%, 80%, or 90% identity, or more, to a polynucleotide sequence that encodes a native Ral2 polypeptide or a portion thereof.
- an immunological fusion partner can be derived from protein D, a surface protein of the gram-negative bacterium Haemophilus 34ittered34 B.
- the immunological fusion partner derived from protein D can be the sequence set forth in SEQ ID NO: 31.
- a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-1 10 amino acids).
- a protein D derivative can be 34ittered34.
- the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes, which can increase the expression level in E. coli and can function as an expression enhancer.
- the lipid tail can ensure optimal presentation of the antigen to antigen presenting cells.
- Other fusion partners can include the non- structural protein from influenza virus, NS 1 (hemagglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes can be used.
- the immunological fusion partner can be the protein known as LYTA, or a portion thereof (particularly a C-terminal portion).
- the immunological fusion partner derived from LYTA can the sequence set forth in SEQ ID NO: 32.
- LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene).
- LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
- the C-terminal domain of the LYTA protein can be responsible for the affinity to the choline or to some choline analogues such as DEAE.
- LYTA E. coli C-LYTA expressing plasmids useful for expression of fusion proteins.
- Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus can be employed.
- a repeat portion of LYTA can be incorporated into a fusion polypeptide.
- a repeat portion can, for example, be found in the C-terminal region starting at residue 178.
- One particular repeat portion can incorporate residues 188-305.
- the target antigen is fused to an immunological fusion partner, which can also be referred to herein as an "immunogenic component,” comprising a cytokine selected from the group of IFN- ⁇ , TNFa, IL-2, IL-8, IL-12, IL- 18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL- 10, IL-13, IL-15, IL-16, IL-17, IL-23, IL-32, M-CSF (CSF- 1), IFN-a, IFN- ⁇ , IL-l a, IL- ⁇ ⁇ , IL-1RA, IL-1 1, IL- 17A, IL-17F, IL- 19, IL-20, IL-21 , IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31 , IL-33, IL-
- the target antigen fusion can produce a protein with substantial identity to one or more of IFN- ⁇ , TNFa IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IL- 16, IL-17, IL-23, IL-32, M-CSF (CSF- 1), IFN-a, IFN- ⁇ , IL-la, IL- ⁇ , IL-1RA, IL-1 1 , IL-17A, IL- 17F, IL-19, IL-20, IL-21 , IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31 , IL-33, IL-34, IL-35, ⁇ -36 ⁇ , ⁇ , ⁇ , IL-36Ra, IL-37, TSLP, LIF, OSM,
- the target antigen fusion can encode a nucleic acid encoding a protein with substantial identity to one or more of IFN- ⁇ , TNFa, IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IL-16, IL- 17, IL-23, IL-32, M-CSF (CSF-1), IFN-a, IFN- ⁇ , IL-la, IL- ⁇ ⁇ , IL-1RA, IL- 1 1 , IL- 17A, IL- 17F, IL- 19, IL-20, IL-21, IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31 , IL-33, IL-34, IL-35, ⁇ -36 ⁇ , ⁇ , ⁇ , IL-36Ra, IL-
- the target antigen fusion further comprises one or more immunological fusion partner, which can also be referred to herein as an "immunogenic components," comprising a cytokine selected from the group of IFN- ⁇ , TNFa, IL-2, IL-8, IL-12, IL- 18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL- 10, IL-13, IL-15, IL-16, IL-17, IL-23, IL-32, M-CSF (CSF- 1), IFN-a, IFN- ⁇ , IL-la, IL- ⁇ ⁇ , IL-1RA, IL-1 1 , IL-17A, IL-17F, IL-19, IL-20, IL-21 , IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31 , IL-33, IL-34, IL
- the sequence of IFN- ⁇ can be, but is not limited to, a sequence as set forth in SEQ ID NO: 33.
- the sequence of TNFa can be, but is not limited to, a sequence as set forth in SEQ ID NO: 34.
- the sequence of IL-2 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 35.
- the sequence of IL-8 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 36.
- the sequence of IL-12 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 37.
- the sequence of IL-18 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 38.
- the sequence of IL-7 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 39.
- the sequence of IL-3 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 40.
- the sequence of IL-4 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 41.
- the sequence of IL-5 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 42.
- the sequence of IL-6 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 43.
- the sequence of IL-9 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 44.
- the sequence of IL-10 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 45.
- the sequence of IL-13 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 46.
- the sequence of IL-15 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 47.
- the sequence of IL-16 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 74.
- the sequence of IL- 17 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 75.
- the sequence of IL-23 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 76.
- the sequence of IL-32 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 77.
- the target antigen is fused or linked to an immunological fusion partner, also referred to herein as an "immunogenic component,” comprising a cytokine selected from the group of IFN- ⁇ , TNFa IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL- 10, IL- 13, IL-15, , IL- 16, IL- 17, IL-23, IL-32, M-CSF (CSF- 1), IFN-a, IFN- ⁇ , IL-l a, IL- ⁇ ⁇ , IL-IRA, IL-1 1 , IL- 17A, IL-17F, IL-19, IL-20, IL-21 , IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL- 30, IL-31 , IL-33, IL-31 , IL-33
- the target antigen is co-expressed in a cell with an immunological fusion partner, also referred to herein as an "immunogenic component," comprising a cytokine selected from the group of IFN- ⁇ , TNFa IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL- 6, IL-9, IL- 10, IL-13, IL-15, IL- 16, IL-17, IL-23, IL-32, M-CSF (CSF- 1), IFN-a, IFN- ⁇ , IL-l a, IL- 1 ⁇ , IL- IRA, IL- 1 1 , IL- 17A, IL- 17F, IL-19, IL-20, IL-21 , IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31 , IL-33,
- the target antigen is fused or linked to an immunological fusion partner, comprising CpG ODN (e.g., Class A, B, or C CpG ODNs; non- limiting examples sequences are shown in SEQ ID NO: 108 - SEQ ID NO: 1 19 in which phosphodiester bases are in capital letters, phosphorothioate bases are in lower case letters, and palindromes are underlined and the colon denotes the reflection point), cholera toxin (a non- limiting example sequence is shown in SEQ ID NO: 49), a truncated A subunit coding region derived from a bacterial ADP-ribosylating exotoxin (a non-limiting example sequence is shown in (a non-limiting example sequence is shown in SEQ ID NO: 50), a truncated B subunit coding region derived from a bacterial ADP-ribosylating exotoxin (a non-limiting example sequence is shown in SEQ ID NO: 50), a
- the target antigen is fused or linked to an immunological fusion partner, comprising an IL-15 superagonist.
- Interleukin 15 IL- 15
- IL- 15 is a naturally occurring inflammatory cytokine secreted after viral infections. Secreted IL- 15 can carry out its function by signaling via its cognate receptor on effector immune cells, and thus, can lead to overall enhancement of effector immune cell activity.
- IL- 15 Based on IL- 15 's broad ability to stimulate and maintain cellular immune responses, it is believed to be a promising immunotherapeutic drug.
- major limitations in clinical development of IL- 15 can include low production yields in standard mammalian cell expression systems and short serum half-life.
- the IL- 15:IL- 15R ⁇ x complex comprising proteins co- expressed by the same cell, rather than the free IL-15 cytokine, can be responsible for stimulating immune effector cells bearing IL- 15 ⁇ receptor.
- IL-15N72D novel IL-15 superagonist mutant
- IL-15N72D additive of either mouse or human IL-15R and Fc fusion protein (the Fc region of immunoglobulin) to equal molar concentrations of IL- 15N72D can provide a further increase in IL- 15 biologic activity, such that IL-15N72D:IL-15Ra/Fc super-agonist complex exhibits a median effective concentration (EC50) for supporting IL-15 -dependent cell growth that was greater thanlO-fold lower than that of free IL- 15 cytokine.
- EC50 median effective concentration
- the IL-15 superagonist can be a novel IL- 15 superagonist mutant (IL- 15N72D).
- addition of either mouse or human IL-15Ra and Fc fusion protein (the Fc region of immunoglobulin) to equal molar concentrations of IL-15N72D can provide a further increase in IL-15 biologic activity, such that IL-15N72D:IL-15R /Fc super-agonist complex exhibits a median effective concentration (EC50) for supporting IL-15-dependent cell growth that can be greater than 10-fold lower than that of free IL- 15 cytokine
- EC50 median effective concentration
- the present disclosure provides a IL- 15N72D:IL- 15Ra/Fc superagonist complex with an EC50 for supporting IL- 15 -dependent cell growth that is greater than 2-fold lower, greater than 3-fold lower, greater than 4-fold lower, greater than 5-fold lower, greater than 6- fold lower, greater than 7-fold lower, greater than 8-fold lower, greater than 9-fold lower, greater than 10-fold lower, greater than 15-fold lower, greater than 20-fold lower, greater than 25-fold lower, greater than 30-fold lower, greater than 35-fold lower, greater than 40-fold lower, greater than 45- fold lower, greater than 50-fold lower, greater than 55-fold lower, greater than 60-fold lower, greater than 65-fold lower, greater than 70-fold lower, greater than 75-fold lower, greater than 80-fold lower, greater than 85-fold lower, greater than 90-fold lower, greater than 95-fold lower, or greater than 100-fold lower than that of free IL-15 cytokine.
- the IL- 15 super agonist is a biologically active protein complex of two IL- 15N72D molecules and a dimer of soluble IL- 15Ra Fc fusion protein, also known as ALT-803.
- ALT-803 a dimer of soluble IL- 15Ra Fc fusion protein
- a soluble fusion protein can be generated by linking the human IL- 15RaSu domain (amino acids 1 -65 of the mature human IL- 15Ra protein) with the human IgGl CH2-CH3 region containing the Fc domain (232 amino acids).
- This IL- 15RctSu/IgG l Fc fusion protein can have the advantages of dimer formation through disulfide bonding via IgG l domains and ease of purification using standard Protein A affinity chromatography methods.
- ALT-803 can have a soluble complex consisting of 2 protein subunits of a human IL- 15 variant associated with high affinity to a dimeric IL- 15Ra sushi domain/human IgGl Fc fusion protein.
- the IL- 15 variant is a 1 14 amino acid polypeptide comprising the mature human IL- 15 cytokine sequence with an Asn to Asp substitution at position 72 of helix C N72D).
- the human IL- 15R sushi domain/human IgG l Fc fusion protein comprises the sushi domain of the IL- 15R subunit (amino acids 1 - 65 of the mature human IL- 15Ra protein) linked with the human IgG l CH2-CH3 region containing the Fc domain (232 amino acids). Aside from the N72D substitution, all of the protein sequences are human.
- the calculated molecular weight of the complex comprising two IL- 15N72D polypeptides is shown in SEQ ID NO: 71
- a disulfide linked homodimeric IL- 15RaSu/IgG l Fc protein is shown in SEQ ID NO: 72
- SEQ ID NO: 72 a disulfide linked homodimeric IL- 15RaSu/IgG l Fc protein
- a recombinant vector encoding for a target antigen and for ALT-803 can have any sequence described herein to encode for the target antigen and can have SEQ ID NO: 71 , SEQ ID NO: 71 , SEQ ID NO: 72, and SEQ ID NO: 72, in any order, to encode for ALT-803.
- Each IL- 15N720 polypeptide can have a calculated molecular weight of approximately 12.8 kDa and the IL- 15RaSu/IgG 1 Fc fusion protein can have a calculated molecular weight of approximately 33.4 kDa.
- Both the IL- 15N72D and IL- 15RaSu/IgG 1 Fc proteins can be glycosylated resulting in an apparent molecular weight of ALT- 803 of approximately 1 14 kDa by size exclusion chromatography.
- the isoelectric point (pi) determined for ALT-803 can range from approximately 5.6 to 6.5.
- the fusion protein can be negatively charged at pH 7.
- Combination therapy with Ad5[E l -, E2b-] vectors encoding for a Chikungunya virus antigen and ALT-803 can result in boosting the immune response, such that the combination of both therapeutic moieties acts to synergistically boost the immune response more than either therapy alone.
- combination therapy with an Ad5[El-, E2b-] vector encoding for a Chikungunya virus antigen and ALT-803 can result in synergistic enhancement of stimulation of antigen-specific effector CD4+ and CD8+ T cells, stimulation of NK cell response directed towards killing infected cells, stimulation of neutrophils or monocyte cell responses directed towards killing infected cells via antibody dependent cell-mediated cytotoxicity (ADCC), or antibody dependent cellular phagocytosis (ADCP) mechanisms.
- ADCC antibody dependent cell-mediated cytotoxicity
- ADCP antibody dependent cellular phagocytosis
- Combination therapy with an Ad5[El-, E2b-] vector encoding for a Chikungunya virus antigen and ALT-803 can synergistically boost any one of the above responses, or a combination of the above responses, to vastly improve survival outcomes after administration to a subject in need thereof.
- any of the immunogenicity enhancing agents described herein can be fused or linked to a target antigen by expressing the immunogenicity enhancing agents and the target antigen in the same recombinant vector, using any recombinant vector described herein.
- SEQ ID NO: 30 SNSRRRSLRWSWLLSVLAAVGLGLATAPAQAAPPALSQDRFADFP
- the nucleic acid sequences for the target antigen and the immunological fusion partner are not separated by any nucleic acids.
- a nucleic acid sequence that encodes for a linker can be inserted between the nucleic acid sequence encoding for any target antigen described herein and the nucleic acid sequence encoding for any immunological fusion partner described herein.
- the protein produced following immunization with the viral vector containing a target antigen, a linker, and an immunological fusion partner can be a fusion protein comprising the target antigen of interest followed by the linker and ending with the immunological fusion partner, thus linking the target antigen to an immunological fusion partner that increases the immunogenicity of the target antigen of interest via a linker.
- the sequence of linker nucleic acids can be from about 1 to about 150 nucleic acids long, from about 5 to about 100 nucleic acids along, or from about 10 to about 50 nucleic acids in length.
- the nucleic acid sequences can encode one or more amino acid residues.
- the amino acid sequence of the linker can be from about 1 to about 50, or about 5 to about 25 amino acid residues in length. In some embodiments, the sequence of the linker comprises less than 10 amino acids. In some embodiments, the linker can be a polyalanine linker, a polyglycine linker, or a linker with both alanines and glycines.
- the adenovirus vectors can be used in a number of vaccine settings for generating an immune response against one or more alphavirus target antigens as described herein, especially chikungunya virus target antigens.
- the adenovirus vectors are of particular importance because of the finding that they can be used to generate immune responses in subjects who have preexisting immunity to adenovirus or adenovirus vectors and can be used in vaccination regimens that include multiple rounds of immunization using the adenovirus vectors, regimens not possible using previous generation adenovirus vectors.
- generating an immune response can comprise an induction of a humoral response and/or a cell-mediated response.
- generating an immune response or “inducing an immune response” can comprise any statistically significant change, e.g., increase in the number of one or more immune cells (T cells, B cells, antigen-presenting cells, dendritic cells, neutrophils, and the like) or in the activity of one or more of these immune cells (CTL activity, HTL activity, cytokine secretion, change in profile of cytokine secretion, etc.).
- Illustrative methods useful in this context can include intracellular cytokine staining (ICS), ELISpot, proliferation assays, cytotoxic T cell assays including chromium release or equivalent assays, and gene expression analysis using any number of polymerase chain reaction (PCR) or RT-PCR based assays.
- ICS cytokine staining
- ELISpot ELISpot
- proliferation assays cytotoxic T cell assays including chromium release or equivalent assays
- gene expression analysis using any number of polymerase chain reaction (PCR) or RT-PCR based assays a number of polymerase chain reaction (PCR) or RT-PCR based assays.
- generating an immune response comprises an increase in target antigen-specific CTL activity of about 1.5 to 20 or more fold, at least, about, or at most 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or any range or number derived therefrom in a subject administered the adenovirus vectors as compared to a control.
- generating an immune response comprises an increase in target-specific CTL activity of about 1.5 to 20, or more fold in a subject administered the adenovirus vectors as compared to a control.
- generating an immune response that comprises an increase in target antigen- specific cell mediated immunity activity as measured by ELISpot assays measuring cytokine secretion, such as interferon-gamma (IFN- ⁇ ), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF- a), granzyme, or other cytokines, of about 1.5 to 20, or more fold as compared to a control.
- cytokine secretion such as interferon-gamma (IFN- ⁇ ), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF- a), granzyme, or other cytokines
- generating an immune response comprises an increase in target- specific antibody production of between 1.5 and 5 fold in a subject administered the adenovirus vectors as compared to an appropriate control. In another embodiment, generating an immune response comprises an increase in target-specific antibody production of about 1.5 to 20, or more fold in a subject administered the adenovirus vector as compared to a control.
- certain aspects can provide methods for generating an immune response against an alphavirus target antigen of interest comprising administering to the subject an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) nucleic acids encoding the target antigen; and readministering the adenovirus vector at least once to the subject; thereby generating an immune response against the target antigen.
- Certain other aspects can provide methods for generating an immune response against alphavirus target antigens of interest comprising administering to the subject an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) nucleic acids encoding the target antigens; and readministering the adenovirus vector at least once to the subject; thereby generating an immune response against the target antigens.
- the vector administered is not a gutted vector.
- methods for generating an immune response against an alphavirus virus target antigen in a subject, wherein the subject has pre-existing immunity to Ad, by administering to the subject an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) nucleic acids encoding the target antigen; and re-administering the adenovirus vector at least once to the subject; thereby generating an immune response against the alphavirus virus target antigen.
- methods can be provided for generating an immune response against alphavirus virus target antigens in a subject, wherein the individual has pre-existing immunity to Ad, by administering to the subject an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) nucleic acids encoding the target antigens; and re-administering the adenovirus vector at least once to the subject; thereby generating an immune response against the alphavirus virus target antigens.
- preexisting immunity to Ad this can be determined using methods known in the art, such as antibody-based assays to test for the presence of Ad antibodies. Further, in certain embodiments, the methods can include first determining that an individual has preexisting immunity to Ad then administering the E2b deleted adenovirus vectors as described herein.
- the adenovirus vector can comprise nucleic acid sequences that encode one or more target antigens of interest from any one or more of the infectious agents against which an immune response is to be generated.
- a target antigen can include, but is not limited to, viral antigen protein, such as E3ALPHA, E2ALPHA, 6K, E I ALPHA, nsP l , nsP2, nsP3, and nsP4.
- the adenovirus vector stock can be combined with an appropriate buffer, physiologically acceptable carrier, excipient or the like.
- an appropriate number of adenovirus vector particles are administered in an appropriate buffer, such as, sterile PBS.
- solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
- a E2b deleted adenovirus vector can be delivered in pill form, delivered by swallowing or by suppository.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
- Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants.
- the prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it can include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- one dosage can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences' ' 15 th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations can need to meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biology standards.
- the carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
- the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
- pharmaceutically-acceptable can refer to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
- compositions described herein, as well as dosage will vary from individual to individual, and from disease to disease, and can be readily established using standard techniques.
- the pharmaceutical compositions and vaccines can be administered by injection (e.g., intracutaneous, intraperitoneal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration), in pill form (e.g., swallowing, suppository for vaginal or rectal delivery).
- injection e.g., intracutaneous, intraperitoneal, intramuscular, intravenous or subcutaneous
- intranasally e.g., by aspiration
- pill form e.g., swallowing, suppository for vaginal or rectal delivery.
- between 1 and 3 doses can be administered over a 6 week period and further booster vaccinations can be given periodically thereafter.
- the replication defective adenovirus is administered at a dose that suitable for effecting an immune response as described herein. In some embodiments, the replication defective adenovirus is administered at a dose from about l xl O 8 virus particles to about 5xl 0 13 virus particles per immunization. In some cases, the replication defective adenovirus is administered at a dose that is from about lxl O 9 to about 5x10 12 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about lxl O 8 virus particles to about 5x10 8 virus particles per immunization.
- the replication defective adenovirus is administered at a dose from about 5xl 0 8 virus particles to about l xl O 9 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about l xlO 9 virus particles to about 5xl0 9 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 5xl 0 9 virus particles to about l xl O 10 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about lxlO 10 virus particles to about 5x10 10 virus particles per immunization.
- the replication defective adenovirus is administered at a dose from about 5xl 0 10 virus particles to about l xl O 1 1 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about l xl O 1 1 virus particles to about 5x10" virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 5x10" virus particles to about lxl O 12 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about lxl O 12 virus particles to about 5x10 12 virus particles per immunization.
- the replication defective adenovirus is administered at a dose from about 5x l 0 12 virus particles to about lxl O 13 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about l xl O 13 virus particles to about 5xl0 13 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about lxl O 8 virus particles to about 5xl0 10 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about lxl O 10 virus particles to about 5xl 0 12 virus particles per immunization.
- the replication defective adenovirus is administered at a dose from about lxl O 1 1 virus particles to about 5xl 0 13 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about l x l O 8 virus particles to about lxl O 10 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about l xlO 10 virus particles to about l xl O 12 virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about lxlO 1 1 virus particles to about 5xl0 13 virus particles per immunization.
- the replication defective adenovirus is administered at a dose that is greater than or equal to lxlO 9 , 2 xl O 9 , 3 xl O 9 , 4 xl O 9 , 5 xlO 9 , 6 xlO 9 , 7 xl O 9 , 8 xlO 9 , 9 xl O 9 , lxlO 10 , 2 xlO'°, 3 xlO 10 , 4 xlO 10 , 5 xlO 10 , 6 xlO 10 , 7 xlO 10 , 8 xlO 10 , 9 xlO 10 , 1 xlO 1 1 , 2 xlO", 3 xlO 1 1 , 4 xlO" , 5x10" , 6 xlO 1 1 , 7 xlO 1 1 , 8 xlO 1 1 , 9 xlO 1 1 , lxlO 12 , 1.5 xl
- the replication defective adenovirus is administered at a dose that is less than or equal to lxlO 9 , 2 xlO 9 , 3 xlO 9 , 4 xlO 9 , 5 xlO 9 , 6 xlO 9 , 7 xlO 9 , 8 xlO 9 , 9 xlO 9 , lxlO 10 , 2 xlO 10 , 3 xlO 10 , 4 xlO 10 , 5 xlO 10 , 6 xlO 10 , 7 xlO 10 , 8 xlO 10 , 9 xl0 10 , 1 xlO 1 1 , 2 xlO 1 1 , 3 xlO 1 1 , 4 xlO 1 1 , 5x10' 6 xlO 1 1 , 7 xlO 1 ', 8 xlO 1 1 , 9 xlO" , lxlO 12 , 1.5 x
- a suitable dose can be an amount of an adenovirus vector that, when administered as described above, is capable of promoting a target antigen immune response as described elsewhere herein.
- the immune response is at least 10-50% above the basal (i.e., untreated) level.
- Such response can be monitored by measuring the target antigen antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing alphavirus infected cells in vitro, or other methods known in the art for monitoring immune responses.
- an appropriate dosage regimen provides the adenovirus vectors in an amount sufficient to provide prophylactic benefit.
- Protective immune responses can generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which can be performed using samples obtained from a patient before and after immunization (vaccination).
- the adenovirus vaccines can also be administered as part of a prime and boost regimen.
- a mixed modality priming and booster inoculation scheme can result in an enhanced immune response.
- one aspect is a method of priming a subject with a plasmid vaccine, such as a plasmid vector comprising a target antigen of interest, by administering the plasmid vaccine at least one time, allowing a predetermined length of time to pass, and then boosting by administering the adenovirus vector.
- a plasmid vaccine such as a plasmid vector comprising a target antigen of interest
- Multiple primings e.g., 1-3, can be employed, although more can be used.
- the length of time between priming and boost can vary from about six months to a year, but other time frames can be used.
- kits of the present disclosure can further comprise instructions regarding the dosage and/or administration regimen information.
- kits comprises a composition and method for providing a vaccine as described herein.
- kits can further comprise components useful in administering the kit components and instructions on how to prepare the components.
- the kit can further comprise software for conducting monitoring of patients before and after vaccination with appropriate laboratory tests, or communicating results and patient data with medical staff.
- the components of the kit can be in dry or liquid form. If they are in dry form, the kit can include a solution to solubilize the dried material.
- the kit can also include transfer factor in liquid or dry form. In some embodiments, if the transfer factor is in dry form, the kit includes a solution to solubilize the transfer factor.
- the kit can also include containers for mixing and preparing the components.
- the kit can also include instrument for assisting with the administration such as, for example, needles, tubing, applicator, inhalant, syringe, pipette, forceps, measured spoon, eye dropper, or any such medically approved delivery vehicle.
- the kits or drug delivery systems as described herein also can include a means for containing compositions of the present disclosure in close confinement for commercial sale and distribution.
- Ad5 [E1 -, E2b-]-CHIKV vaccine is an adenovirus serotype 5 (Ad5) vector that was modified by removal of El , E2b, and E3 gene regions, and insertion of a CHIKV gene.
- the nucleic acid sequence encoding a CHIKV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 2) including CHIKV structural proteins C, E3, E2, 6K, and E l was cloned into an Ad5 [E 1 -, E2b-]-based platform to produce Ad5 [E1 -, E2b-]- CHIKVs t r using a homologous recombination-based approach.
- Ad5 [E1 -, E2b-]-CHIKV str was produced in E.C7 cells (FIG. 2).
- the replication-deficient virus was propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 62ittered.
- Viral infectious titer was determined as plaque-forming units (PFUs) on an E.C7 cell monolayer.
- the VP concentration was determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.
- This example illustrates the results of injection of an Ad5 [E 1-, E2b-]-CHIKV str for generating protective immunity against CHIKV infection.
- mice Groups of ten ( 10) mice each were immunized two times subcutaneously at weekly intervals with a dose of 10 9 VPs Ad5 [E1-, E2b-]-CHIKV str .
- Control mice were injected with PBS. Mice were then challenged with a lethal dose of CHIKV by injection of the virus into the footpad.
- Control mice succumbed to infection within 7 days (FIG. 4A), lost weight (FIG. 4B), and demonstrated prolonged inflammation at the footpad (FIG. 4C).
- vaccinated mice survived CHIKV infection (FIG. 4A), did not lose weight (FIG. 4B), and demonstrated rapid resolution of inflammation of the footpad (FIG. 4C).
- This example illustrates construction of an Ad5 [E1-, E2b-] vector containing multiple CHIKV antigens.
- Ad5 [E1 -, E2b-] containing multiple CHIKV genes three individual alphavirus antigen gene sequences ((1) C, E3, E2, 6K, and El gene ((nucleotides 7567- 1 1313 of SEQ ID NO: 1); (2) NS gene 1 ; (3) NS gene 2) are separated by "self-c leaving" 2A peptide derived from Porcine teschovir s- 1 and Thosea asigna virus respectively (FIG. 5A) (de Felipe P and Ryan M Traffic 2004 5(8), 616-26; Hoist J et al. Nature Immunol. 2008 9 :658-66 ; Kim JH et al.
- This example illustrates the construction of Ad5 [E1-, E2b-] vectors containing an ONNV antigen, and testing for expression and protective immunity by multiple injections of the vectors.
- Ad5 [E1 -, E2b-]-ONNV str vaccine is an adenovirus serotype 5 (Ad5) vector that is modified by removal of El , E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding an ONNV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 5) using a homologous recombination- based approach.
- ONNV structural proteins are expressed as a result of infection of E.C7 cells with Ad5 [E1 -, E2b-]-ONNV str . ONNV structural protein expression is detected by western blot with an anti-ONNV antibody.
- mice Groups of ten (10) mice each are immunized two times subcutaneously at weekly intervals with a dose of 10 9 VPs Ad5 [E1-, E2b-]-ONNV str .
- Control mice are injected with PBS or 10 9 VPs Ad5-null.
- Mice are then challenged with a lethal dose of ONNV by injection of the virus. The survival rate, immune response, and weight are compared between the mice injected with the vaccine and the control group.
- This example illustrates the construction of Ad5 [E1-, E2b-] vectors containing an MAYV antigen and testing for expression and protective immunity by multiple injection of the vectors.
- Ad5 [E 1 -, E2b-]-MAYV str vaccine is an adenovirus serotype 5 (Ad5) vector that is modified by removal of El , E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding an MAYV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 8) using a homologous recombination-based approach.
- Ad5 [E 1 -, E2b-]-MAYV str is produced in E.C7 cells.
- the replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 64ittered.
- Viral infectious titer is determined as plaque-forming units (PFUs) on an E.C7 cell monolayer.
- the VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.
- MAYV structural proteins are expressed as a result of infection of E.C7 cells with Ad5 [E1 -, E2b-]-MAYV str .
- MAYV structural protein expression is detected by western blot with an anti- MAYV antibody.
- This example illustrates the construction of Ad5 [E1-, E2b-] vectors containing a RRV antigen and testing for expression and protective immunity by multiple injection of the vectors.
- Ad5 [E1 -, E2b-]-RRV str vaccine is an adenovirus serotype 5 (Ad5) vector that is modified by removal of El , E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding a RRV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 1 1) using a homologous recombination-based approach.
- Ad5 [E 1 -, E2b-]-RRV str is produced in E.C7 cells. The replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 65ittered.
- Viral infectious titer is determined as plaque-foiming units (PFUs) on an E.C7 cell monolayer.
- the VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 run and 280 nm.
- mice Groups of ten (10) mice each are immunized two times subcutaneously at weekly intervals with a dose of 10 9 VP Ad5 [E 1 -, E2b-]-RRV str .
- Control mice are injected with PBS or 10 9 VPs Ad5- null.
- Mice are then challenged with a lethal dose of RRV by injection of the virus. The survival rate, immune response, and weight are compared between the mice injected with the vaccine and the control group.
- This example illustrates the construction of Ad5 [E1 -, E2b-] vectors containing a VEEV antigen and testing for expression and protective immunity by multiple injection of the vectors.
- Ad5 [E 1 -, E2b-]-VEEV str vaccine is an adenovirus serotype 5 (Ad5) vector that is modified by removal of E l , E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding a VEEV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 14) using a homologous recombination-based approach.
- Ad5 adenovirus serotype 5
- Ad5 [E 1 -, E2b-]-VEEV s t r is produced in E.C7 cells.
- the replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 65ittered.
- Viral infectious titer is determined as plaque-forming units (PFUs) on an E.C7 cell monolayer.
- the VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.
- mice Groups of ten (10) mice each are immunized two times subcutaneously at weekly intervals with a dose of 10 9 VP Ad5 [E1-, E2b-]-VEEV str .
- Control mice are injected with PBS or 10 9 VPs Ad5-null.
- Mice are then challenged with a lethal dose of VEEV by injection of the virus. The survival rate, immune response, and weight are compared between the mice injected with the vaccine and the control group.
- Ad5 [E1 -, E2b-]-EEEV str vaccine is an adenovirus serotype 5 (Ad5) vector that is modified by removal of El , E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding an EEEV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 17) using a homologous recombination-based approach.
- Ad5 adenovirus serotype 5
- Ad5 [E1-, E2b-]-EEEV str is produced in E.C7 cells.
- the replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 66ittered.
- Viral infectious titer is determined as plaque-forming units (PFUs) on an E.C7 cell monolayer.
- the VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.
- EEEV structural proteins are expressed as a result of infection of E.C7 cells with Ad5 [E1 -, E2b-]-EEEV slr . EEEV structural protein expression is detected by western blot with an anti-EEEV antibody.
- Ad5 [E1 -, E2b-]-WEEV str vaccine is an adenovirus serotype 5 (Ad5) vector that has been modified by removal of El , E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding a WEEV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 20) using a homologous recombination-based approach.
- Ad5 [E 1-, E2b-]-WEEV str is produced in E.C7 cells.
- the replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 66ittered.
- Viral infectious titer is determined as plaque-forming units (PFUs) on an E.C7 cell monolayer.
- the VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.
- WEEV structural proteins are expressed as a result of infection of E.C7 cells with Ad5 [E 1 -, E2b-]-WEEV str . WEEV structural protein expression is detected by western blot with an anti-WEEV antibody.
- mice Groups of ten (10) mice each are immunized two times subcutaneously at weekly intervals with a dose of 10 9 VPs Ad5 [E 1-, E2b-]-WEEV str .
- Control mice are injected with PBS or 10 9 VPs Ad5-null.
- Mice are then challenged with a lethal dose of WEEV by injection of the virus. The survival rate, immune response, and weight are compared between the mice injected with the vaccine and the control group.
- This example illustrates pre-clinical studies of Ad5 [E1 -, E2b-]-Chikungunya vaccines in mice, including assessment of cell mediate immune (CMI) responses, cytolytic T lymphocyte (CTL) responses, intracellular cytokine expression, and antibody secretion.
- CMI cell mediate immune
- CTL cytolytic T lymphocyte
- Pre-clinical studies included administration of Ad5 [E1 -, E2b-]-Chikungunya vaccines, a comparison to controls, and an assessment of the immune responses in mice.
- FIG. 6 illustrates CMI responses (IFN- ⁇ ) and CTL responses and Granzyme B responses in splenocytes from immunized or control mice using an ELISpot assay.
- C57BL/6 mice were immunized two times at two-week intervals with lxlO 10 VPs Ad5 [E1-, E2b-]- CHIK vaccine comprising SEQ ID NO: 1 or with 1X10 10 VPs Ad5 [E 1 -, E2b-]-null (empty vector controls).
- splenocytes from individual mice were tested for induction of immune responses after exposure of cells to Chikungunya virus peptides.
- the data show the cumulative number of spot forming cells (SFCs) per 10 6 splenocytes after exposure to three separate pools of Chikungunya virus peptides (peptide numbers for Chikungunya were large enough to merit division into three separate pools to use in assays - CHIKV peptide pool 1 comprised peptides 1 -103, CHIKV peptide pool 2 comprising peptides 104-207, and peptide pool 3 comprising peptides 208-310).
- FIG. 6A illustrates CMI responses in Ad5 [E 1-, E2b- ]-CHIK immunized and control C57BL/6 mice as measured by IFN- ⁇ secreting SFCs using an ELISpot assay. Specificity of the response is evidenced by the lack of reactivity of splenocytes to the negative control SIV-nef peptide pool. A high number of IFN- ⁇ secreting SFCs were induced in Ad5 [E1-, E2b-]-CHIK immunized mice as compared to control mice.
- FIG. 6A illustrates CMI responses in Ad5 [E 1-, E2b- ]-CHIK immunized and control C57BL/6 mice as measured by IFN- ⁇ secreting SFCs using an ELISpot assay. Specificity of the response is evidenced by the lack of reactivity of splenocytes to the negative control SIV-nef peptide pool. A high number of IFN- ⁇ secreting SFCs were induced in Ad5 [E1
- FIG. 6B illustrates CTL responses in Ad5 [E1 -, E2b-]-CHIK immunized and control C57BL/6 mice as measured by Granzyme B (Gr- B) secreting SFCs using an ELISpot assay. Specificity of the response is evidenced by the lack of reactivity of splenocytes to the negative control SIV-nef peptide pool. A high number of Granzyme B secreting SFCs were induced in Ad5 [E 1 -, E2b-]-CHIK immunized mice as compared to control mice.
- Gr- B Granzyme B
- FIG. 7 illustrates lymphocyte activation in splenocytes from immunized or control C57BL/6 mice as measured by intracellular expression of IFN- ⁇ or IFN-y/TNF- ⁇ analyzed by flow cytometry.
- C57BL/6 mice were immunized two times at two-week intervals with lxlO 10 VPs Ad5 [E1-, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with lxlO 10 VPs Ad5 [E1-, E2b-]-null (empty vector controls).
- splenocytes from individual mice were exposed to three separate pools of Chikungunya virus peptides (peptide numbers for Chikungunya were large enough to merit division into three separate pools to use in assays - CHIKV peptide pool 1 comprised peptides 1 - 103, CHIKV peptide pool 2 comprising peptides 104-207, and peptide pool 3 comprising peptides 208-310) and analyzed by flow cytometry for induction of intracellular cytokine expression.
- FIG. 7A illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN- ⁇ in CD8+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools). A higher percentage of CD8+ splenocytes from immunized mice expressed intracellular IFN- ⁇ as compared to negative controls.
- FIG. 7B illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN- ⁇ in CD4+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools). A higher percentage of CD4+ splenocytes from immunized mice expressed intracellular IFN- ⁇ as compared to negative controls.
- FIG. 7C illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN- ⁇ and TNF-a in CD8+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools). A higher percentage of CD8+ splenocytes from immunized mice expressed intracellular IFN- ⁇ and TNF-a as compared to negative controls.
- FIG. 7D illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN- ⁇ and TNF-a in CD4+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools). A higher percentage of CD4+ splenocytes from immunized mice expressed intracellular IFN- ⁇ and TNF-a as compared to negative controls.
- FIG. 8 illustrates anti-Chikungunya envelope protein-2 antibody responses in immunized mice as compared to control mice as measured by a quantitative enzyme-linked immunosorbent assay (ELISA).
- C57BL/6 mice were immunized two times at two-week intervals with l xl O 10 VPs Ad5 [E 1-, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with lxl O 10 VPs Ad5 [E 1-, E2b-]-null (empty vector controls).
- mice were evaluated for induction of antibody responses. Blood was collected by cheek pouch laceration under anesthesia. Anti-Chikungunya specific antibody responses were induced at higher levels in immunized mice as compared to control mice.
- This example illustrates prevention of Chikungunya infection by prophylaxis with any Ad5 [E1 -, E2b-] -Chikungunya vaccine of this disclosure including an Ad5 [E1 -, E2b-] with any one of, or any combination of, Chikungunya antigens (e.g., SEQ ID NO: 1 - SEQ ID NO: 3) inserted into the adenovirus vector.
- An Ad5 [E1 -, E2b-] -Chikungunya vaccine is constructed as described in EXAMPLE 1 for a single-targeted Chikungunya vaccine or is constructed as described in EXAMPLE 3 for a multi-targeted Chikungunya vaccine.
- the Ad5 [E1-, E2b-]-Chikungunya vaccine is administered to a subject subcutaneously, intradermal ly, or intramuscularly, once or every two weeks for a total of two immunizations.
- Cellular and humoral immune responses against Chikungunya virus and protection against infection by Chikungunya virus is induced after immunization of a subject with the Ad5 [E1-, E2b-] -Chikungunya vaccine.
- immunity by prophylaxis with the Ad5 [E1 -, E2b-]-Chikungunya vaccine is conferred to the subject.
- the subject is any animal including a human, a non-human primate, or any other non-human animal.
- This example illustrates prevention of Chikungunya infection by prophylaxis with any Ad5 [E 1-, E2b-] -Chikungunya vaccine of this disclosure including an Ad5 [E1 -, E2b-] with any one of, or any combination of, Chikungunya antigens (e.g., SEQ ID NO: 1 - SEQ ID NO: 3) inserted into the adenovirus vector in combination with any co-stimulatory molecule described herein.
- any Ad5 [E 1-, E2b-] -Chikungunya vaccine of this disclosure including an Ad5 [E1 -, E2b-] with any one of, or any combination of, Chikungunya antigens (e.g., SEQ ID NO: 1 - SEQ ID NO: 3) inserted into the adenovirus vector in combination with any co-stimulatory molecule described herein.
- Chikungunya antigens e.g., SEQ ID NO: 1
- An Ad5 [E1-, E2b-] -Chikungunya vaccine is constructed as described in EXAMPLE 1 for a single-targeted Chikungunya vaccine or is constructed as described in EXAMPLE 3 for a multi-targeted Chikungunya vaccine.
- the Ad5 [E1-, E2b-] -Chikungunya vaccine is administered subcutaneously, intradermally, or intramuscularly to a subject once or every two weeks for a total of two immunizations.
- the Ad vaccine is co-administered with a co-stimulatory molecule, such as a tolllike receptor (TLR) agonist mixed with the vaccine formulation.
- TLR tolllike receptor
- This example illustrates prevention of Chikungunya infection by prophylaxis with any Ad5 [E1-, E2b-]-CHIK vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, Chikungunya antigens (e.g., SEQ ID NO: 1 - SEQ ID NO: 3) inserted into the adenovirus vector as well as any immunological fusion partner described herein, also encoded by the adenovirus vector.
- any Ad5 [E1-, E2b-]-CHIK vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, Chikungunya antigens (e.g., SEQ ID NO: 1 - SEQ ID NO: 3) inserted into the adenovirus vector as well as any immunological fusion partner described herein, also encoded by the adenovirus vector.
- Chikungunya antigens e.
- An Ad5 [E 1 -, E2b-]-CHIK vaccine is constructed as described in EXAMPLE 1 for a single-targeted Chikungunya vaccine or is constructed as described in EXAMPLE 3 for a multi-targeted Chikungunya vaccine with the Ad vector additionally encoding for any immunological fusion partner disclosed herein.
- the Ad5 [E1-, E2b-]-Chikungunya vaccine is administered subcutaneously, intradermally, or intramuscularly to a subject, once or every two weeks for a total of two immunizations.
- Vaccines with immunological fusion partners are administered subcutaneously, intradermally, or intramuscularly.
- This example illustrates prevention of o'nyong-nyong virus (ONNV) infection by prophylaxis with any Ad5 [E1-, E2b-]- ONNV vaccine of this disclosure including an Ad5 [E 1 -, E2b-] with any one of, or any combination of, ONNV antigens (e.g., SEQ ID NO: 4 - SEQ ID NO: 6) inserted into the adenovirus vector.
- An Ad5 [E1-, E2b-]-ONNV vaccine is constructed as adapted from EXAMPLE 1 or as described in EXAMPLE 4 for a single-targeted ONNV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted ONNV vaccine.
- the Ad5 [E1-, E2b- ]-ONNV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations.
- Cellular and humoral immune responses against ONNV virus and protection against infection by ONNV virus is induced after immunization of a subject with the Ad5 [E 1-, E2b-]- ONNV vaccine.
- immunity by prophylaxis with the Ad5 [E1-, E2b-]-ONNV vaccine is conferred to the subject.
- the subject is any animal including a human, a non-human primate, or any other non-human animal.
- This example illustrates prevention of Ross River virus (RRV) infection by prophylaxis with any Ad5 [E 1 -, E2b-]-RRV vaccine of this disclosure including an Ad5 [E 1-, E2b-] with any one of, or any combination of, RRV antigens (e.g., SEQ ID NO: 10 - SEQ ID NO: 12) inserted into the adenovirus vector.
- An Ad5 [E1 -, E2b-]-RRV vaccine is constructed as adapted from EXAMPLE 1 or described in EXAMPLE 6 for a single-targeted RRV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted RRV vaccine.
- the Ad5 [E1 -, E2b-]-RRV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations.
- Cellular and humoral immune responses against RRV virus and protection against infection by RRV virus is induced after immunization of a subject with the Ad5 [E1-, E2b-]- RRV vaccine.
- immunity by prophylaxis with the Ad5 [E1-, E2b-]-RRV vaccine is conferred to the subject.
- the subject is any animal including a human, a non-human primate, or any other non-human animal.
- This example illustrates prevention of Marayo fever virus (MAYV) infection by prophylaxis with any Ad5 [E1 -, E2b-]-MAYV vaccine of this disclosure including an Ad5 [E 1 -, E2b-] with any one of, or any combination of, MAYV antigens (e.g., SEQ ID NO: 7 - SEQ ID NO: 9) inserted into the adenovirus vector.
- An Ad5 [E1 -, E2b-]-MAYV vaccine is constructed as adapted from EXAMPLE 1 or described in EXAMPLE 5 for a single-targeted MAYV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted MAYV vaccine.
- the Ad5 [E 1-, E2b-]-MAYV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations.
- Cellular and humoral immune responses against MAYV virus and protection against infection by MAYV virus is induced after immunization of a subject with the Ad5 [E 1 -, E2b-]-MAYV vaccine.
- immunity by prophylaxis with the Ad5 [E 1-, E2b-]-MAYV vaccine is conferred to the subject.
- the subject is any animal including a human, a non-human primate, or any other non-human animal.
- the Ad5 [E1 -, E2b-]-VEEV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations.
- Cellular and humoral immune responses against VEEV virus and protection against infection by VEEV virus is induced after immunization of a subject with the Ad5 [E1 -, E2b-]-VEEV vaccine. .
- immunity by prophylaxis with the Ad5 [E 1-, E2b-]-VEEV vaccine is conferred to the subject.
- the subject is any animal including a human, a non-human primate, or any other non-human animal.
- This example illustrates prevention of Western equine encephalomyelitis virus (WEEV) infection by prophylaxis with any Ad5 [E1 -, E2b-]-WEEV vaccine of this disclosure including an Ad5 [E 1 -, E2b-] with any one of, or any combination of, WEEV antigens (e.g., SEQ ID NO: 19 - SEQ ID NO: 21) inserted into the adenovirus vector.
- An Ad5 [E1 -, E2b-]-WEEV vaccine is constructed as adapted from EXAMPLE 1 or described in EXAMPLE 9 for a single-targeted WEEV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted WEEV vaccine.
- the Ad5 [E1 -, E2b-]-WEEV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations.
- Cellular and humoral immune responses against WEEV virus and protection against infection by WEEV virus is induced after immunization of a subject with the Ad5 [E 1-, E2b-]-WEEV vaccine.
- immunity by prophylaxis with the Ad5 [E1 -, E2b-]-WEEV vaccine is conferred to the subject.
- the subject is any animal including a human, a non-human primate, or any other non-human animal.
- This example illustrates prevention of Eastern equine encephalitis virus (EEEV) infection by prophylaxis with any Ad5 [E1-, E2b-]-EEEV vaccine of this disclosure including an Ad5 [E1 -, E2b-] with any one of, or any combination of, EEEV antigens (e.g., SEQ ID NO: 16 - SEQ ID NO: 18) inserted into the adenovirus vector.
- An Ad5 [E1 -, E2b-]-EEEV vaccine is constructed as adapted from EXAMPLE 1 or described in EXAMPLE 8 for a single-targeted EEEV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted EEEV vaccine.
- the Ad5 [E1 -, E2b-]- EEEV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations.
- Cellular and humoral immune responses against EEEV virus and protection against infection by EEEV virus is induced after immunization of a subject with the Ad5 [E1-, E2b-]- EEEV vaccine.
- immunity by prophylaxis with the Ad5 [E1 -, E2b-]-EEEV vaccine is conferred to the subject.
- the subject is any animal including a human, a non-human primate, or any other non-human animal.
- the Ad5 [E 1-, E2b-]-alphavirus vaccine is comprised of any of combination of: single-targeted or multi-targeted Ad5 [El, E2b]-ON V vector as described in EXAMPLE 15, single-targeted or multi-targeted Ad5 [El , E2b]-CHIK vector as described in EXAMPLE 11, single-targeted or multi-targeted Ad5 [El, E2b]-RRV vector as described in EXAMPLE 16, single-targeted or multi-targeted Ad5 [El , E2b]-MAYV vector as described in EXAMPLE 17, single-targeted or multi-targeted Ad5 [El , E2b]-VEEV vector as described in EXAMPLE 18, single-targeted or multi-targeted Ad5 [El , E2b]-WEEV vector as described in EXAMPLE 18, single-targeted or multi-targeted Ad5 [El , E2b]-WEEV vector as described in EXAMP
- the Ad5 [E 1-, E2b-]-alphavirus vaccine is comprised of an Ad5 [E1-, E2b-] with any combination of at least two antigens from different alphaviruses inserted into the adenovirus vector.
- the at least two antigens is comprised of any combination of an ONNV antigen (e.g., any one of SEQ ID NO: 4 - SEQ ID NO: 6), a CHIK antigen (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 3), an EEEV antigen (e.g., any one of SEQ ID NO: 16 - SEQ ID NO: 18), a WEEV antigen (e.g., any one of SEQ ID NO: 19 - SEQ ID NO: 21), a VEEV antigen (e.g., any one of SEQ ID NO: 13 - SEQ ID NO: 15), a MAYV antigen (e.g., any one of SEQ ID NO: 7 - SEQ ID NO: 9), and/or a R V antigen (e.g., any one of SEQ ID NO: 10 - SEQ ID NO: 12).
- an ONNV antigen e.g., any one of SEQ ID NO: 4 - SEQ
- the Ad5 [E1 -, E2b-] -alphavirus vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations.
- Cellular and humoral immune responses against alphaviruses and protection against infection by alphavirus is induced after immunization of a subject with the Ad5 [E 1-, E2b-]-alphavirus vaccine.
- immunity by prophylaxis with the Ad5 [E1 -, E2b-]-alphavirus vaccine is conferred to the subject.
- the subject is any animal including a human, a non-human primate, or any other non-human animal.
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Abstract
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AU2017297610A AU2017297610B2 (en) | 2016-07-15 | 2017-07-14 | Compositions and methods for alphavirus vaccination |
KR1020197004334A KR20190031266A (ko) | 2016-07-15 | 2017-07-14 | 알파바이러스 백신접종을 위한 조성물 및 방법 |
CA3030451A CA3030451C (fr) | 2016-07-15 | 2017-07-14 | Compositions et procedes pour la vaccination contre les alphavirus |
EP17828595.3A EP3484508A4 (fr) | 2016-07-15 | 2017-07-14 | Compositions et procédés pour la vaccination contre les alphavirus |
US16/317,747 US11104916B2 (en) | 2016-07-15 | 2017-07-14 | Compositions and methods for alphavirus vaccination |
JP2019501689A JP6903120B2 (ja) | 2016-07-15 | 2017-07-14 | アルファウイルスワクチン接種のための組成物及び方法 |
CN201780054911.4A CN109803677A (zh) | 2016-07-15 | 2017-07-14 | 用于α病毒疫苗接种的组合物和方法 |
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US201662363136P | 2016-07-15 | 2016-07-15 | |
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PCT/US2017/042272 WO2018014008A1 (fr) | 2016-07-15 | 2017-07-14 | Compositions et procédés pour la vaccination contre les alphavirus |
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US (1) | US11104916B2 (fr) |
EP (1) | EP3484508A4 (fr) |
JP (1) | JP6903120B2 (fr) |
KR (1) | KR20190031266A (fr) |
CN (1) | CN109803677A (fr) |
AU (1) | AU2017297610B2 (fr) |
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WO2019016756A1 (fr) * | 2017-07-21 | 2019-01-24 | Glaxosmithkline Biologicals Sa | Constructions d'antigène du virus chikungunya |
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WO2020143634A1 (fr) * | 2019-01-07 | 2020-07-16 | 康希诺生物股份公司 | Vaccin à arnsam et procédé de préparation associé |
WO2020231855A1 (fr) * | 2019-05-10 | 2020-11-19 | Nant Holdings Ip, Llc | Nogapendékine alfa-inbakicept pour thérapies de stimulation immunitaire et traitement d'infections virales |
WO2021099906A1 (fr) * | 2019-11-18 | 2021-05-27 | Janssen Biotech, Inc. | Vaccins basés sur les mutants du gène calr et de la protéine jak2 et leurs utilisations |
WO2024059501A1 (fr) * | 2022-09-15 | 2024-03-21 | University Of Florida Research Foundation, Inc. | Dosages pour la détection de virus mayaro et leurs procédés de détection |
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BR112019018915A2 (pt) | 2017-03-15 | 2020-04-14 | Pandion Therapeutics Inc | imunotolerância direcionada |
MX2019013517A (es) | 2017-05-24 | 2020-08-17 | Pandion Operations Inc | Inmunotolerancia dirigida. |
US10174092B1 (en) | 2017-12-06 | 2019-01-08 | Pandion Therapeutics, Inc. | IL-2 muteins |
US10946068B2 (en) | 2017-12-06 | 2021-03-16 | Pandion Operations, Inc. | IL-2 muteins and uses thereof |
CN109536464B (zh) * | 2018-12-10 | 2022-06-10 | 中国科学院武汉病毒研究所 | 一种缺失衣壳蛋白基因的基孔肯雅病毒感染性克隆及构建方法和在制备减毒疫苗中的应用 |
TW202110885A (zh) | 2019-05-20 | 2021-03-16 | 美商潘迪恩治療公司 | 靶向MAdCAM之免疫耐受性 |
EP4107187A4 (fr) | 2020-02-21 | 2024-07-03 | Pandion Operations Inc | Immunotolérance ciblée sur un tissu avec un effecteur cd39 |
US11857620B2 (en) * | 2020-03-11 | 2024-01-02 | Immunitybio, Inc. | Method of inducing immunity against SARS-CoV-2 using spike (s) and nucleocapsid (N)-ETSD immunogens delivered by a replication-defective adenovirus |
KR20230031756A (ko) * | 2021-08-27 | 2023-03-07 | 한국과학기술원 | 신종 코로나바이러스 감염증 대응 예방 백신 조성물 |
CN117964722A (zh) * | 2024-01-31 | 2024-05-03 | 扬州优邦生物药品有限公司 | 一种预防猪盖他病毒的病毒样颗粒疫苗及其制备方法 |
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- 2017-07-14 AU AU2017297610A patent/AU2017297610B2/en active Active
- 2017-07-14 EP EP17828595.3A patent/EP3484508A4/fr active Pending
- 2017-07-14 CA CA3030451A patent/CA3030451C/fr active Active
- 2017-07-14 US US16/317,747 patent/US11104916B2/en active Active
- 2017-07-14 CN CN201780054911.4A patent/CN109803677A/zh active Pending
- 2017-07-14 KR KR1020197004334A patent/KR20190031266A/ko not_active Application Discontinuation
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- 2017-07-14 JP JP2019501689A patent/JP6903120B2/ja active Active
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Also Published As
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EP3484508A1 (fr) | 2019-05-22 |
JP2019521148A (ja) | 2019-07-25 |
AU2017297610B2 (en) | 2020-03-12 |
US11104916B2 (en) | 2021-08-31 |
AU2017297610A1 (en) | 2019-01-31 |
CN109803677A (zh) | 2019-05-24 |
CA3206542A1 (fr) | 2018-01-18 |
CA3030451A1 (fr) | 2018-01-18 |
EP3484508A4 (fr) | 2020-03-25 |
KR20190031266A (ko) | 2019-03-25 |
CA3030451C (fr) | 2023-09-05 |
JP6903120B2 (ja) | 2021-07-14 |
US20200172925A1 (en) | 2020-06-04 |
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